The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses

Abstract

Abstract Background Sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol. Cholesterol is a membrane component and is a precursor of ecdysteroids, but cannot be synthesized de novo in insects. However, a direct association between SCPx gene expression, cholesterol absorption and development in lepidopteran insects remains to be experimentally demonstrated. Results An SCPx cDNA (Sl SCPx) cloned from the common cutworm, Spodoptera litura, was characterized. The Sl SCPx cDNA encoded a 535-amino acid protein consisting of a 3-oxoacyl-CoA thiolase (SCPx-t) domain and a SCP-2 (SCPx-2) domain. Sl SCPx mRNA was expressed predominately in the midgut, while Sl SCPx-2 mRNA was detected in the midgut, fat body and epidermis and no Sl SCPx-t mRNA was detected. A 58-kDa full-length SCPx protein and a 44-kDa SCPx-t protein were detected in the midgut of sixth instar larvae when the anti-Sl SCPx-t antibody was used in western blotting analysis; a 16-kDa SCP-2 protein was detected when anti-Sl SCPx-2 antibody was used. Sl SCPx protein was post-translationally cleaved into two smaller proteins, SCPx-t and SCPx-2. The gene appeared to be expressed into two forms of mRNA transcripts, which were translated into the two proteins, respectively. Sl SCPx-t and Sl SCPx-2 proteins have distinct and different locations in the midgut of sixth instar larvae. Sl SCPx and Sl SCPx-t proteins were detected predominately in the cytoplasm, whereas Sl SCPx-2 protein was detected in the cytoplasm and nuclei in the Spli-221 cells. Over-expression of Sl SCPx and Sl SCPx-2 proteins enhanced cholesterol uptake into the Spli-221 cells. Knocking-down Sl SCPx transcripts by dsRNA interference resulted in a decrease in cholesterol level in the hemolymph and delayed the larval to pupal transition. Conclusion Spatial and temporal expression pattern of this Sl SCPx gene during the larval developmental stages of S. litura showed its specific association with the midgut at the feeding stage. Over-expression of this gene increased cholesterol uptake and interference of its transcript decreased cholesterol uptake and delayed the larval to pupal metamorphosis. All of these results taken together suggest that this midgut-specific Sl SCPx gene is important for cholesterol uptake and normal development in S. litura.